The invention relates to nucleic acids and encoded polypeptides of the human zinc finger protein RIP60. The invention also relates to isolated nucleic acid molecules, expression vectors containing those molecules and host cells transfected with those molecules.
The ability to transfer nucleic acids into cells has vast experimental and therapeutic implications. Many different chemical, electrochemical and biological approaches have been used for this purpose. In vitro chemical methods include osmotic shock transformation of prokaryotic cells and calcium phosphate transfection and liposome-mediated transfer for eukaryotic cells. Nucleic acids, namely DNA, have also been delivered to cells by electroporation. While this latter approach is amenable to nucleic acid transfer in vitro, it is inherently unsuitable for in vivo use. Biological approaches have focused on viral strategies which include retroviral and most recently adenoviral mediated gene transfer into cells in culture and, in some instances, cells in vivo. A common disadvantage of the above-mentioned strategies is their inability to specifically target cells for nucleic acid delivery. Targeting of cell subsets usually requires the selective harvesting of cells followed by in vitro delivery and re-introduction in vivo.
Viral mediated gene transfer requires the in vitro production of defective viral particles which encapsulate a nucleic acid of a finite size. The encapsulated nucleic acid, usually referred to as a viral vector, is a recombinant nucleic acid which contains a gene(s) of interest cloned between 5xe2x80x2 and 3xe2x80x2 flanking viral cis elements. The cis elements are required for integration into the host genome yet they are also capable of transcriptional regulation. As a result, these elements have the potential to interfere with the transcriptional activity of the cloned gene(s). Another limitation of viral mediated gene transfer is the need for and the difficulty in achieving high titre viral stocks. In vivo infection with viruses, when applicable, is generally not effective given the in vivo dilution of viral particles. Additionally, although both retroviral and adenoviral methods employ replication-defective viral particles, the possibility of producing replication-competent viruses and thereby causing active infection in vivo is an inherent danger of both systems.
For retroviral mediated gene transfer to occur, target cells whether in vitro or in vivo must be in a cycling status. Since retroviruses package nucleic acid in the form of RNA, reverse transcription of the RNA to DNA is required for integration into the host genome from where the gene exerts its effects. Cells which divide infrequently or never at all, such as some classes of stem cells or terminally differentiated end cells, are usually less amenable to gene transfer via retroviral infection as compared to rapidly dividing cells. Thus diseases for which a long-term cure is dependent upon stem cell or end cell manipulation are poor candidates for gene therapy treatment using retroviral transfection. Retroviral use is also limited to the restricted range of host infectivity specific to each strain of virus. In contrast adenoviruses which contain double stranded DNA do not require target cells to be cycling for infection, integration and propagation.
DNA has also been delivered to cells using receptor-mediated endocytosis. In this approach, DNA is initially complexed with polycations such as polylysine for condensation and charge neutralization purposes. Ligands for cell surface receptors, such as transferrin, are then coupled either biochemically or enzymatically to the polylysine moieties. In a further modification, the transferrin molecules are coupled to the outer surface of inactivated adenoviral particles. The adenoviral particles can effect the release of the DNA/polylysine/transferrin complex from endosomes prior to lysosome mediated degradation. The transfer of up to 48 kilobases (kb) of DNA has been reported using this approach. Cotten et al., PNAS v. 89, p.6094-6098 (1992).
In contrast to the use of polycations for complexing DNA, other approaches have incorporated specific DNA binding domains which recognize and bind distinct nucleic acid consensus sequences. An example of this is the use of the GAL4 DNA binding domain of yeast which selectively binds to a 17 bp sequence. Thus a nucleic acid to be delivered must usually be modified to incorporate artificial GAL4 binding sites. Likewise, other approaches which rely on a consensus sequence dependent DNA binding domain will similarly require modification of the transferred nucleic acid.
The invention also relates to the molecular cloning and characterization of RIP60, a zinc finger protein involved in cell division and nucleic acid replication.
The invention provides isolated RIP60 nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated RIP60 polypeptides, and agents which bind RIP60 polypeptides, including antibodies.
According to one aspect of the invention, isolated nucleic acid molecules are provided that comprise: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:50 and which code for a polypeptide having RIP60 activity, (b) deletions, additions and substitutions of (a) which code for a polypeptide having RIP60 activity, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule comprises SEQ ID NO: 1. In other embodiments, the isolated nucleic acid molecule comprises SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:50. In some embodiments, the isolated nucleic acid molecules are those that code for a polypeptide comprising SEQ ID NO:2. In some embodiments, the isolated nucleic acid molecules are those that code for a polypeptide comprising SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:51. In an important embodiment, the nucleic acid molecules code for a native RIP60 polypeptide, including homologs and alleles. A native RIP60 polypeptide is one which possesses a native RIP60 function or activity, such as but not limited to DNA binding or protein multimerization. Another function or activity of a native RIP60 polypeptide is the ability to bind to either itself or to other proline rich region containing proteins, specifically through its proline rich region.
The invention in another aspect provides an isolated nucleic acid molecule selected from the group consisting of (a) a unique fragment of nucleic acid molecule of SEQ ID NO:1 of sufficient length to represent a sequence unique within the human genome, and (b) complements of (a), provided that the unique fragment includes a sequence of contiguous nucleotides which is not identical to a sequence selected from the sequence group consisting of (1) sequences having the GenBank and EMBL database accession numbers of Table 1, (2) complements of (1), and (3) fragments of (1) and (2).
In one embodiment, the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, and (6) at least seven contiguous nucleotides nonidentical to the sequence group.
In another embodiment, the fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and every integer length therebetween as if fully cited herein.
In other embodiments, the unique fragment encodes a peptide which is a fragment of a polypeptide consisting of SEQ ID NO:2.
According to another aspect, the invention provides expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is encoded by the foregoing isolated nucleic acid molecules of the invention. In important embodiments, the isolated polypeptide is encoded by the nucleic acid of SEQ ID NO:1, giving rise to a xcx9c62 kD polypeptide having the sequence of SEQ ID NO:2 that can bind to nucleic acids, preferably at ATT-rich regions and even more preferably at USR and DSR sequences, and form multimers on such nucleic acids. In certain embodiments, the isolated polypeptide is a polypeptide having RIP60 activity. Preferably, the polypeptide is a native RIP60 polypeptide. In important embodiments, the isolated polypeptide comprises SEQ ID NO:2. In still other embodiments, the isolated polypeptide comprises SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:51.
In other embodiments, an isolated peptide is provided which comprises a fragment or variant of the foregoing polypeptides of sufficient length to represent a sequence unique within the human genome, and to identify a polypeptide having RIP60 activity or, in other embodiments, a native RIP60 polypeptide. The isolated peptide may comprise at least 6, at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 16, at least 18, or at least 20 contiguous amino acids having a sequence of a fragment of SEQ ID NO:2. Isolated peptides which are immunogenic are also provided.
According to another aspect of the invention, compositions are provided which comprise an isolated agent that binds selectively to a polypeptide having RIP60 activity, including a native RIP60 polypeptide, encoded by the foregoing isolated nucleic acid molecules of the invention. Preferably, the isolated agent binds selectively to a polypeptide comprising SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:51, or to a fragment thereof. In important embodiments, the isolated agent is a peptide. In a further embodiment, the peptide is an antibody or a fragment thereof (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to a polypeptide having RIP60 activity). In even more preferred embodiments, the antibody is a humanized antibody or a chimeric antibody. The isolated agent may be conjugated to a detectable label. The detectable label may be selected from the group consisting of a radioactive label, an enzyme, a biotin molecule, an avidin molecule or a fluorochrome.
In yet another aspect, the invention relates to a kit comprising a package including an agent that selectively binds the isolated nucleic acid molecules and polypeptides of the invention, a control for comparing with a measured or test value, instructions and optionally related materials. In a further embodiment, the kit contains a control which has a predetermined value for comparing to the measured or test value.
Another aspect of the invention is a method for determining the level of RIP60 expression in a sample from a subject. RIP60 expression is defined either as RIP60 mRNA expression or RIP60 polypeptide expression. Various methods can be used to measure expression. Preferred embodiments of the invention include PCR and Northern blotting for measuring RIP60 mRNA expression, and monoclonal or polygonal antisera to RIP60 as reagents to measure RIP60 polypeptide expression. In certain embodiments, test samples are biopsy samples, and biological fluids such as blood. The method involves measuring a test level of RIP60 expression in a test sample and comparing the test level of RIP60 expression to a control.
Each of the limitations of the invention can encompass various embodiments of the invention. It is therefore anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.
SEQ ID NO:1 is the nucleotide sequence of human RIP60 cDNA.
SEQ ID NO:2 is the amino acid sequence of human RIP60 polypeptide.
SEQ ID NO:3 is the nucleotide sequence of the Z2 domain of the human RIP60.
SEQ ID NO:4 is the amino acid sequence of the Z2 domain of the human RIP60.
SEQ ID NO:5 is the nucleotide sequence of the proline rich region of human RIP60.
SEQ ID NO:6 is the amino acid sequence of the proline rich region of human RIP60.
SEQ ID NO:7 is the nucleotide sequence of the primer p512.
SEQ ID NO:8 is the nucleotide sequence of the primer p520.
SEQ ID NO:9 is the nucleotide sequence of the primer p 521.
SEQ ID NO:10 is the nucleotide sequence of the primer OCH7.
SEQ ID NO:11 is the nucleotide sequence of the primer OCH8.
SEQ ID NO:12 is the nucleotide sequence of the primer OCH13.
SEQ ID NO:13 is the nucleotide sequence of the primer OCH14.
SEQ ID NO:14 is the nucleotide sequence of the primer OCH35.
SEQ ID NO:15 is the nucleotide sequence of the primer OCH36.
SEQ ID NO:16 is the nucleotide sequence of the primer OCH37.
SEQ ID NO:17 is the nucleotide sequence of the primer OCH38.
SEQ ID NO:18 is the nucleotide sequence of the primer OCH39.
SEQ ID NO:19 is the nucleotide sequence of the primer OCH40.
SEQ ID NO:20 is the nucleotide sequence of the primer RIP1.
SEQ ID NO:21 is the nucleotide sequence of the primer RIP2.
SEQ ID NO:22 is the nucleotide sequence of the primer RIP3.
SEQ ID NO:23 is the nucleotide sequence of the primer RIP4.
SEQ ID NO:24 is the nucleotide sequence of the primer RIP5.
SEQ ID NO:25 is the nucleotide sequence of the primer RIP6.
SEQ ID NO:26 is the nucleotide sequence of the primer RIP7.
SEQ ID NO:27 is the nucleotide sequence of the primer RIP8.
SEQ ID NO:28 is the nucleotide sequence of the primer RIP9.
SEQ ID NO:29 is the nucleotide sequence of the primer RIP10.
SEQ ID NO:30 is the amino acid sequence of a tryptic fragment from RIP60.
SEQ ID NO:31 is the amino acid sequence of a tryptic fragment from RIP60.
SEQ ID NO:32 is the amino acid sequence of RIP60 zinc finger 1.
SEQ ID NO:33 is the amino acid sequence of RIP60 zinc finger 2.
SEQ ID NO:34 is the amino acid sequence of RIP60 zinc finger 3.
SEQ ID NO:35 is the amino acid sequence of RIP60 zinc finger 4.
SEQ ID NO:36 is the amino acid sequence of RIP60 zinc finger 5.
SEQ ID NO:37 is the amino acid sequence of RIP60 zinc finger 6.
SEQ ID NO:38 is the amino acid sequence of RIP60 zinc finger 7.
SEQ ID NO:39 is the amino acid sequence of RIP60 zinc finger 8.
SEQ ID NO:40 is the amino acid sequence of RIP60 zinc finger 9.
SEQ ID NO:41 is the amino acid sequence of RIP60 zinc finger 10.
SEQ ID NO;42 is the amino acid sequence of RIP60 zinc finger 11.
SEQ ID NO:43 is the amino acid sequence of RIP60 zinc finger 12.
SEQ ID NO:44 is the amino acid sequence of RIP60 zinc finger 13.
SEQ ID NO:45 is the amino acid sequence of RIP60 zinc finger 14.
SEQ ID NO:46 is the amino acid sequence of RIP60 zinc finger 15.
SEQ ID NO:47 is the amino acid consensus sequence for RIP60 zinc fingers.
SEQ ID NO:48 is the nucleotide sequence of the Z1 domain of RIP60.
SEQ ID NO:49 is the amino acid sequence of the Z1 domain of RIP60.
SEQ ID NO:50 is the nucleotide sequence of the Z2 and the PRR domain of RIP60.
SEQ ID NO:51 is the amino acid sequence of the Z2 and the PRR domain of RIP60.
SEQ ID NO:52 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:53 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:54 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:55 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:56 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:57 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:58 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:59 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:60 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:61 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:62 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:63 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:64 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:65 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:66 is the nucleotide sequence of a molecule which Z2 binds.
SEQ ID NO:67 is the nucleotide sequence of the DSR site.
SEQ ID NO:68 is the amino acid sequence of the proline rich region of RIP60 and adjacent regions.